Exogenous RNA can be delivered into cells by way of vectorization, for example using natural polycationic peptides termed protamine. The inventor has previously shown that under specific conditions protamine and RNA can form nanoparticles, termed protamine-RNA particles, which can deliver RNA into cells, particularly into cell compartments (e.g. endosomes) where immune sensors of infections, such as Toll-Like Receptors (TLRs) 7 and 8 are located (Rettig L. et al., 2010, Blood 115(22):4533-41 and WO 2009/144230 A1). The particles may activate TLR-7 when taken up by, for example, plasmacytoid dendritic cells, or TLR-8 when taken up by, for example, monocytes. In addition, at best when they are smaller than 450 nm, the particles can deliver their RNA content into the cytosol and thereby allow, for example, protein expression in case the RNA entrapped in the protamine particles is a coding RNA, such as messenger RNA (mRNA).
However, the present inventor observed that the protamine-RNA nanoparticles, particularly those below 450 nm, are unstable, e.g. enlarge over time, when diluted in salty pharmaceutical carriers, such as 0.9% saline, Ringer or Ringer Lactate. As a consequence, they lose their physical characteristics (e.g. size) and associated biological properties (e.g. specific immunostimulating features or cytosolic release of the entrapped RNA) when formulated for injection, i.e. in isotonic salty solution.
Accordingly, it was an object of the present invention to provide aqueous formulations of particles comprising a polycation, such as protamine, and RNA that are characterized by an increased physical stability of the particles contained therein and can be used for injection.